The gut endoderm gives rise to the major cell types of many internal organs, including the thyroid, thymus, lung, stomach, liver, pancreas, intestine and bladder. A rigorous understanding of normal gut endoderm morphogenesis, including knowledge of the origin, commitment, specification and differentiation of cells generating gut endoderm and its derivative tissues, should underpin logical efforts to understand disease progression and design new therapeutic strategies for these vital organ systems. The prevailing view of germ layer formation in mammalian embryos is that ectoderm, mesoderm and gut endoderm derive solely from the epiblast during gastrulation and while extraembryonic tissues interact with the epiblast to establish the body axes, they contribute solely to extraembryonic structures, such as the yolk sac and placenta. The cell movements underlying the morphogenesis of the gut endoderm, the tissue that gives rise to the respiratory and digestive tracts and associated organs such as the lungs, liver and pancreas, are complex and not well understood. Using live imaging combined with genetic labeling, we have begun to investigate the role of the visceral endoderm, a presumed extraembryonic tissue, in gut endoderm formation in the mouse (Figure 1). Three key findings arise from our preliminary studies. (1) A new model for gut endoderm morphogenesis in the mouse, incorporating a novel morphogenetic mechanism resulting in extensive mixing of embryonic and extraembryonic cells. (2) The stereotypical congregation of extraembryonic (visceral) endoderm cells around signaling centers of the gastrula stage embryo. (3) The segregation of extraembryonic and embryonic tissues, a fundamental feature of mammalian development, may not be as strict as believed, and the visceral endoderm, a lineage defined as exclusively extraembryonic, may contribute cells to the embryo-proper. These questions will be explored in the following three Specific Aims. Figure 1: Combining live imaging and genetics in mice to investigate gut endoderm formation in mammals. Live imaging and genetic labeling of the visceral endoderm, and its derivatives in mouse embryos. At embryonic day (E) 6.5 the entire visceral endoderm is labeled with a green fluorescent protein (GFP) reporter. GFP protein reveals a dispersed population of visceral endoderm-derived cells present in the embryonic region of the conceptus at E7.5, which by E8.75 become incorporated into the gut tube of the embryo (A,B). Specific Aim 1: To elucidate the cell behaviors that mediate gut endoderm morphogenesis. We will use both existing and new reporter strains to visualize cell dynamics in wild type embryos and mutants that exhibit endoderm defects. Specific Aim 2: To determine the sequence of events leading to the organization of extraembryonic (visceral) endoderm cells around signaling centers of the mouse gastrula, and test the hypothesis that this arrangement is central to the function of these signaling centers. This will be achieved using live imaging and the analysis of mutants that fail to generate midline structures. Specific Aim 3: To determine if a lineage relationship exists between the visceral endoderm and the gut endoderm tissues of the fetus and adult mouse. We will establish if the visceral endoderm is a transient or stable component of embryonic and adult gut endoderm derived tissues. We will test the hypothesis that extraembryonic (visceral) endoderm is not an exclusively extraembryonic tissue using a genetic induced fate mapping approach. PUBLIC HEALTH RELEVANCE: The gut endoderm gives rise to the major cell types of many internal organs, including the thyroid, thymus, lung, stomach, liver, pancreas, intestine and bladder. A rigorous understanding of normal gut endoderm morphogenesis, including knowledge of the origin, commitment, specification and differentiation of cells generating gut endoderm and its derivative tissues, should underpin logical efforts to design new therapeutic strategies for this vital organ system. The prevailing view of germ layer formation in mammalian embryos is that ectoderm, mesoderm and gut endoderm derive solely from the pluripotent epiblast during gastrulation, and while extraembryonic tissues interact with the epiblast to establish the body axes, they contribute solely to extraembryonic structures, such as the yolk sac and placenta. Our preliminary studies challenge this view, and inform the hypotheses to be tested through the Specific Aims of this proposal. Three key findings arise from our preliminary studies in mice. (1) A new model for gut endoderm formation in the mouse, incorporating a novel morphogenetic mechanism resulting in extensive mixing of embryonic and extraembryonic cells. (2) The stereotypical congregation of extraembryonic (visceral) endoderm cells around signaling centers of the gastrula stage embryo. (3) The segregation of extraembryonic and embryonic tissues, a fundamental feature of mammalian development, may not be as strict as believed, and the visceral endoderm, a lineage defined as exclusively extraembryonic, may contribute cells to the embryo-proper. The broad aim of this project is to use a combination of molecular, embryological and live imaging techniques to investigate the role played by the visceral endoderm, a presumed extraembryonic lineage, in the morphogenesis of the mammalian gut endoderm, the tissue that will give rise to the respiratory and digestive tracts, and associated organs such as lungs, liver and pancreas. In Aim 1, we will define the cell behaviors that mediate gut endoderm morphogenesis in the embryo. We will use both existing and new reporter strains to visualize cell dynamics in wild type embryos and mutants that exhibit endoderm defects. In Aim 2, we will determine the sequence of events leading to the organization of extraembryonic (visceral) endoderm cells around signaling centers of the mouse gastrula, and test the hypothesis that this arrangement is central to the function of these signaling centers. This will be achieved using live imaging and the analysis of mutants that fail to generate midline structures. In Aim 3, we will determine if a lineage relationship exists between the visceral endoderm and the gut endoderm tissues of the fetus and adult mouse. We will establish if the visceral endoderm is a transient or stable component of embryonic and adult gut endoderm derived tissues, and will test the hypothesis that extraembryonic (visceral) endoderm is not an exclusively extraembryonic tissue using a genetic induced fate mapping approach.